Hamilton, Grant Edmond; (1998) Cell engineering for the improved recovery of intracellular products from the yeast Saccharomyces cerevisiae. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Sequence analysis of a clone previously isolated by Stateva et al. (1991) and thought to contain the SRBl gene revealed that the clone contained the PDE2 gene. The PDE2 gene of Saccharomyces cerevisiae, which encodes the high affinity cAMP phosphodiesterase, was subsequently demonstrated to be an extragenic suppressor of the osmotic fragility mutation, srbl-1. Disruption of PDE2 was also shown to confer the phenotype on yeast cells of susceptibility to lysis upon osmotic shock. The isolation and analysis of a gene which, when present in multiple copies, suppresses the osmotic sensitivity of the yeast srbl-1 mutation has suggested a role for elements of the Ras2/cAMP signal transduction pathway in the maintenance of cell wall integrity. Further confirmation of this idea was provided by the observation that srbl-1 mutants had elevated levels of intracellular cAMP, comparable to those of pde2 mutant strains. Protein release from srbl-1 cells upon osmotic shock is due to a proportion of the cell population releasing its total protein content. This lysis ability is greatest with exponentially growing cells (70 mg protein/g dry cell weight). Homogenisation studies revealed that the mechanical strength of the mutants are significantly less than that of the wild-type strain (120 mg/g compared to 37 mg/g of wild-type cells, at 1200 Bar). Interestingly, an osmotic shock prior to homogenisation resulted in a large increase in the rate of cell breakage during all stages of growth. This effect was just as apparent with wild-type cells. Stationary phase cells of the wild-type released only 37 mg/g, following an osmotic shock the same sample yielded 91 mg/g dry cell weight at the same homogenisation pressure. Osmotic shock, as a pretreatment to mechanical cell disruption, may prove useful for protein recovery by reducing the power input required by homogenisation and easing the clarification problems associated with the fine debris produced by excessive homogenisation. This may also prove beneficial if viability and activity of the product are essential as is the case with enzymes.

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